The role of arbuscular mycorrhizal fungi in the transfer of nutrients
between white clover and perennial ryegrass
Rogers, J. B., Laidlaw, A. S., & Christie, P. (2001). The role of arbuscular mycorrhizal fungi in the transfer of
nutrients between white clover and perennial ryegrass. Chemosphere, 42, 153-159.
Published in:
Chemosphere
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Download date:18. Jun. 2017
Chemosphere 42 (2001) 153±159
The role of arbuscular mycorrhizal fungi in the transfer of
nutrients between white clover and perennial ryegrass
Jacqueline B. Rogers
a,1
, A. Scott Laidlaw
a,*
, Peter Christie
b
a
b
Department of Applied Plant Science, The QueenÕs University of Belfast, Newforge Lane, Belfast BT9 5PX, UK
Department of Agricultural and Environmental Science, The QueenÕs University of Belfast, Newforge Lane, Belfast BT9 5PX, UK
Abstract
A glasshouse experiment was conducted in which 15 N was used as a tracer applied as (15 NH4 )2 SO4 to donor plants of
white clover and perennial ryegrass. Nitrogen transfer via hyphae of arbuscular mycorrhizal fungi (AMF) or by other
routes was studied by separating the root systems of the two plant species, as donors and receivers, when growing in the
same pot, with selective mesh barriers of varying pore sizes in the presence and absence of AMF. Inoculation with AMF
increased DM production and nitrogen (N) yield of clover plants. Transfer of 15 N occurred between white clover and
grass plants but was independent of AMF. Pore size of the mesh barriers controlled the degree of 15 N enrichment in the
grass, suggesting that transfer was mediated by mass ¯ow and/or di€usion. Additional experiments showed that grass
roots could pass through pores of 60-lm diameter, and hyphal links could not be detected by autoradiography, thus
supporting the conclusions of the tracer experiment. Ó 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Arbuscular mycorrhiza; Nitrogen transfer; White clover; Perennial ryegrass
1. Introduction
Proposed routes for the transfer of nutrients, especially nitrogen (N), from legume to associated grass include death and decay of nodules and roots (Butler et al.,
1959; Dubach and Russelle, 1994), exudation from
legume roots (Ta et al., 1986) or by hyphal links formed
by arbuscular mycorrhizal fungi (AMF) between legume
and grass roots (Haystead et al., 1988). It is generally
acknowledged that in grass/white clover associations
turnover of N in roots, nodules and stolons is the major
source of transferable N (Laidlaw et al., 1996) but the
evidence for hyphae-mediated transfer of N between
*
Corresponding author.
E-mail address: [email protected] (A. Scott
Laidlaw).
1
Present Address. School of Environmental Management
and Geography, University of the West of England, Frenchay
Campus, Coldharbour Lane, Bristol BS16 1QY, UK.
legumes and grass is con¯icting (Haystead et al., 1988;
Barea et al., 1989; Frey and Sch
uepp, 1993; Ikram et al.,
1994).
The strongest evidence for the positive role of N
transfer by AMF from white clover to grass has been
presented by Haystead et al. (1988) using a split root
technique to label N in white clover. They compared the
amount of N transferred to grass sharing the part of the
clover root system fed from the remainder of the root
system in an adjacent pot in the presence and absence of
AMF. Roots in the shared pot were separated by barriers of mesh, the pore sizes of which were considered to
allow solute di€usion and mass ¯ow (3 lm), hyphae to
pass through (60 lm), or the roots were completely
separated by solid barriers or allowed to interact unhindered. Preliminary studies with mesh barriers of
varying pore size indicated that 60 lm would allow roots
to penetrate (Rogers, 1993) and so raised the possibility
that the e€ect found by Haystead et al., may have been
due to grass roots penetrating the mesh and exploring
the N-rich rhizosphere in the clover compartment.
0045-6535/01/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 4 5 - 6 5 3 5 ( 0 0 ) 0 0 1 2 0 - X
154
J.B. Rogers et al. / Chemosphere 42 (2001) 153±159
Rogers (1993) also found that a mesh with pore size of
5 lm was suciently small to prevent passage of external AMF hyphae. The present study was undertaken to
investigate the e€ectiveness of barriers of varying mesh
sizes in limiting N transfer and to re-evaluate the evidence regarding the role of AMF in forming e€ective
hyphal links between perennial ryegrass and white
clover.
2. Materials and methods
2.1. Experiment 1. The role of AMF in the transfer of
nitrogen from white clover to perennial ryegrass
The method of Haystead et al. (1988) outlined brie¯y
above was followed with some modi®cations. In the split
root assemblages, the barrier used to separate the root
systems of the 15 N-fed donor and the receiver comprised
nylon mesh of pore dimensions 5, 15 or 60 lm in addition to a complete barrier and no barrier.
Inoculum comprised heavily mycorrhizal root fragments and adhering soil from grass/white clover plots in
an experiment already described (Laidlaw et al., 1996).
The inoculum was mixed thoroughly with the sterilised
soil/sand mixture in the appropriate treatments and a
pad of infected roots was also inserted below the roots of
transplanted seedlings.
Each assemblage consisted of two pots. All pots
…15 15 cm2 † were ®lled with three parts autoclaved
acid-washed builderÕs sand: one part c-irradiated (1
Mrad) loam soil from the A horizon of a permanent
pasture and the experiment comprised 60 assemblages.
The experimental design was three replicated blocks of
the ®ve barrier treatments with or without inoculum and
with grass or clover as the `receiver'. One of the pots in
each assemblage contained half of the root system of
each pair of donor plants to which 15 N was applied,
while the other contained the other half of the root
system of the donor plants and that of the pair of receiver plants, separated from the donor roots unless
barrierless. The mesh barriers were glued to 1-mm thick
PVC frames, which in turn were axed to the pots with
plastic cement and sealed with a self-curing non-toxic
rubber sealant. The complete barrier was positioned
with sealant only which was subsequently found to be
inadequate and the e€ectiveness of the complete barrier
broke down.
Plants were grown in a slightly heated glasshouse
(daytime maximum in the absence of sunlight was 15°C)
and natural light was supplemented with mercury vapour lamps supplying 150±400 lmol mÿ2 sÿ1 for 12 h per
day. 50 ml 25% full strength nutrient solution was applied weekly and 50 ml distilled water was applied daily
to each pot containing roots of both species. Three
weeks after setting up the experiment, 3.5 mg
(15 NH4 )2 SO4 at 99.7 at.% excess was applied in solution
every 2±3 days to the pot in each assemblage which
contained half of the donor root systems. A total of 124
mg N per pot was supplied. 11 applications were made
before the ®rst harvest, 16 between the ®rst and second
harvests and 8 between the second and third. Donor and
receiver plants were harvested to 2 cm above soil level 7,
12 and 18 weeks after planting. After 26 weeks donor
shoots were harvested to ground level while receivers
were harvested to 2 cm. Receiver shoots and roots were
destructively sampled after a further four weeks.
The proportion of length of roots infected with AMF
was assessed in samples of roots of receiver plants which
were cleared and stained at the end of the experiment
using a modi®cation of the procedure of Phillips and
Hayman (1970). The proportion was estimated using a
grid-line intersection method from presence or absence
of infection in roots at 200 intersections using 50 root
segments per sample. Harvested herbage was dried at
60°C, milled and passed through a 280-lm sieve and
analysed for total N and 15 N with a Europa Scienti®c
ANCA/MS.
2.2. Experiment 2. To investigate the ability of grass and
clover roots to penetrate mesh with pores of di€erent
diameters
14-day-old seedlings of white clover (cv Huia) and
perennial ryegrass (cv Talbot), germinated on moist ®lter paper, were transplanted into 9-cm Petri dishes to
provide a con®ned space to encourage penetration of the
roots through the mesh of given pore size which lined
the Petri dishes. Each dish had drainage holes on the
base and was lined with a disc (diameter 11 cm) of 60,
35, 15 or 5 lm nylon mesh. The disc allowed the base
and sides of the dish to be lined with a 2.5-mm lip
around the top of the dish. The base of the dish was
®lled with a 1:1 (v/v) sand:soil mixture. The seedling
was planted in the growth medium and the shoot was
threaded through a hole of approximately 3-mm diameter cut in the centre of the lid of the dish. The lid was
sealed to the base with insulating tape. 12 replicates were
set up for each mesh size. The seedlings in the Petri
dishes were arranged on plastic trays and placed in a
growth cabinet at 20°C constant temperature set at a
16-h daylength and 90% humidity. 25 ml water and 10 ml
of four-fold nutrient solution (Dart and Pate, 1959) were
applied to the trays daily, ensuring that nutrients were
always available within the Petri dishes.
Six of the replicates were examined under a dissecting
microscope for penetration of the mesh by seedling roots
after ®ve weeks growth in the cabinet and the remaining
six replicates were examined after a further four weeks.
In instances where roots had penetrated, their length
and diameter and presence or absence of branches was
recorded.
J.B. Rogers et al. / Chemosphere 42 (2001) 153±159
2.3. Experiment 3. To investigate the possibility of
transfer of carbon between roots of mycorrhizal perennial
ryegrass and white clover using 14 C autoradiography
Perennial ryegrass (cv Talbot) and white clover (cv
Huia) seedlings were grown in a glasshouse (conditions
similar to Experiment 1) in seed trays, grass for six
weeks and clover for 8 weeks, in a sterilised sand:soil
mixture either inoculated with AM fungi by mixing with
colonised root fragments and AMF spores or uninoculated. Root washings were applied to the latter in an
attempt to ensure microbial populations (other than
mycorrhizal fungi) were similar in both media. 100-ml
quarter strength nutrient solution was applied weekly
and 100 ml distilled water twice weekly. After 6 weeks
the grass and clover roots were 30±45% and 50±70%
mycorrhizal, respectively.
One clover and one grass plant, either mycorrhizal or
non-mycorrhizal, were transplanted as a pair in factorial
combination into specially adapted 9-cm diameter Petri
dishes ®lled with sterilised sand comprising particles of
0.8±1.0 mm. Three holes of 5 mm diameter were drilled in
the lid of each dish, one at one quarter along a diameter
line, one in the centre and one three quarters along the
same line. A plant was transplanted in the dish at the site
of each of the two outer holes and water and nutrients
were applied through the central hole. Each dish received
10 ml water twice weekly and 10 ml quarter strength
nutrient solution once per week. After 4 weeks the lids of
the dishes were sealed to the base with Terostat.
Shoots of the clover plants were enclosed in a cellophane bag that had a 1-cm square rubber septum sealed
on its surface. This allowed labelled salt solution and
acid to generate 14 C to be introduced inside the bag into
a 10 ml glass vial which had been placed on the lid of the
Petri dish before the bag covered the clover shoots.
14
CO2 was generated by adding excess concentrated
lactic acid to calcium carbonate Ca14 CO3 . This was
calculated to expose the clover to 25 lCi of 14 CO2 . After
36 h of exposure, clover shoots were excised and the root
systems prepared for autoradiography by washing soil
from the roots in a ®ne jet of water but minimising
disturbance by retaining the root system in the Petri
dish. The root systems were photographed at 40 ´
magni®cation through the transparent dish base before
being gently removed from the dish, blotted dry and
placed on autoradiographic paper and sealed within a
wooden cassette for 24 h. Photographs and autoradiographs were compared to identify the organs which had
14
C in the developed autoradiographic plates.
155
control and is not considered further. The 5-lm barrier
is therefore taken as the treatment representing minimum rhizosphere interaction.
Mean infection rate for roots of grass (based on root
length infected) was 27.6% for inoculated and 0% for
uninoculated treatments compared to 35.6% and 1.2%
for corresponding infection rates for clover (Table 1).
AMF inoculation had little e€ect on N content and DM
yield of grass donor plants but signi®cantly increased N
content and DM yield of white clover shoots from
harvest 2 and 15 N enrichment of N in clover donor
shoots was signi®cantly reduced due to AMF inoculation. Inoculation signi®cantly increased clover receiver
shoot DM yield at three of the ®ve harvests but had no
e€ect on grass receiver DM yield (data not presented).
Nitrogen content of clover receiver shoots was signi®cantly increased by inoculation at harvests 2 and 4 but
grass receiver shoots were una€ected by inoculation.
Inoculation did not a€ect 15 N enrichment of grass or
clover receiver shoots (Table 2).
Mean shoot DM yield of clover receiver plants was
lower than grass shoots at the ®rst harvest but higher
than grass at harvests 3, 4 and 5 (Table 3). Barrier type
interacted with species at harvest 1 due to grass shoot
DM being lower in the 5 lm mesh treatment than in the
other treatments (except 15 lm), and at harvest 4 caused
by DM yield of clover shoots being lower in the 60 lm
treatments than the other barrier types. Clover receiver
shoots had consistently higher N content than those of
grass but N content was not a€ected by barrier type
(Table 4). Except for harvest 1, clover receiver shoots
had signi®cantly lower enrichment than grass receiver
shoots. From harvest 3 onwards, enrichment of grass
shoots was increased with increasing mesh pore size
(Table 5).
A mesh of pore size 35 lm allowed a few grass roots
to penetrate, although the mean length of roots which
breached the barriers was less than those which penetrated a 60 lm pore size (Table 6). With both of these
mesh sizes, the mean width of the root at the other side
Table 1
Experiment 1: percentage of root length of receiver plants
colonised by AM fungi at the end of the experiment
Barrier (lm)
Inoculum
Grass
Clover
Total
+
)
+
)
+
)
+
)
+
)
30
0
27
0
33
0
22
0
26
0
42
2
38
1
32
0
43
3
23
0
5
15
3. Results
60
Due to the breakdown of the seams around the solid
barrier, the `total barrier' treatment was an inadequate
No barrier
156
J.B. Rogers et al. / Chemosphere 42 (2001) 153±159
Table 2
Experiment 1: N content (g kgÿ1 ) and at.% excess in herbage in inoculation treatments (mean of barrier treatments) in receiver plant
shoots
Harvest
Receiver
Inoculum
N content (g kgÿ1 )
1
Clover
+
)
+
)
25.9
21.5
15.7
13.4
+
)
+
)
39.5
35.6
15.3
14.3
+
)
+
)
39.4
36.8
17.5
15.9
+
)
+
)
34.2
28.0
12.7
12.0
+
)
+
)
24.6
21.1
18.4
17.1
Grass
2
Clover
Grass
3
Clover
Grass
4
Clover
Grass
5
Clover
Grass
SEM
1.91ns
1.91ns
0.98*
0.98ns
0.57ns
0.57ns
0.58*
0.58ns
0.99ns
0.99ns
of the barrier from the plant was about twice that of the
size of the pores. Clover roots were incapable of penetrating any of the barriers but root hairs were apparent
at the other side of the barrier from the plant in barriers
of 35 and 60 lm pore size.
In Experiment 3, 14 C was detected in roots and
shoots of white clover. Some roots of grass also contained 14 C. Although AMF hyphae could be seen, no
hyphal links were apparent in the autoradiographs.
at.%
15
N excess
2.49
3.88
1.85
1.41
SEM
0.35ns
0.35ns
0.07
0.11
0.21
0.50
0.11ns
0.11ns
0.11
0.08
0.70
0.55
0.09ns
0.09ns
0.02
0.03
1.01
0.82
0.08ns
0.08ns
0.11
0.14
1.18
1.38
0.05ns
0.05ns
4. Discussion
Although mycorrhizal clover had a higher N yield
and lower 15 N at.% excess enrichment than nonmycorrhizal clover, AMF infection did not appear to
increase transfer of N from white clover to grass (or vice
versa). The higher N yield and lower enrichment of
mycorrhizal than non-mycorrhizal clover can be
explained by AMF enhancing N2 -®xation (Hayman,
Table 3
Experiment 1: DM yield (g potÿ1 ) of receiver shoots in each barrier treatment (mean of inoculation treatments)
Species
Barrier (lm)
Harvest
2
3
4
5
Clover
Total
5
15
60
None
328
358
420
368
505
273
610
468
392
448
281
387
471
269
469
415
777
840
497
622
757
1360
1258
1035
1310
Grass
Total
5
15
60
None
1478
518
723
930
888
463
540
463
447
448
705
456
705
534
713
268
168
268
285
297
737
448
737
715
778
Species
Barrier
Species ´ Barrier
SEM
SEM
SEM
1
36.0
57.0
80.6
49.2
77.8
110.0
1.57
2.48
3.51
31.7
50.1
70.9
54.1
85.5
120.9
J.B. Rogers et al. / Chemosphere 42 (2001) 153±159
157
Table 4
Experiment 1: nitrogen concentration (g kgÿ1 ) in herbage in each barrier treatment (mean of inoculation treatments) in receiver plant
shoots
Species
Barrier (lm)
Harvest
1
2
3
4
5
Clover
Total
5
15
60
None
22.2
24.4
24.8
23.5
23.7
37.4
39.9
37.9
34.7
38.0
37.3
38.0
38.9
36.9
39.4
33.4
31.1
30.3
28.5
31.9
23.8
23.1
24.4
21.5
21.7
Grass
Total
5
15
60
None
15.1
13.7
15.9
13.2
14.8
14.6
15.7
14.6
14.0
14.7
16.2
17.2
16.2
16.6
16.7
12.3
12.5
12.3
12.7
11.9
18.4
18.0
18.4
18.5
17.3
Species
Barrier
Species ´ Barrier
SEM
SEM
SEM
Table 5
Experiment 1: at.%
15
0.94
1.49
2.10
0.50
0.79
1.11
0.49
0.78
1.10
0.45
0.71
1.01
0.57
0.90
1.27
N excess in herbage in each barrier treatment (mean of inoculation treatments) in receiver plant shoots
Species
Barrier (lm)
Harvest
1
2
3
4
5
Clover
Total
5
15
60
None
3.21
2.95
3.50
3.60
3.68
0.13
0.06
0.11
0.10
0.05
0.08
0.15
0.08
0.10
0.05
0.03
0.02
0.02
0.02
0.02
0.21
0.08
0.10
0.23
0.12
Grass
Total
5
15
60
None
1.18
1.84
2.22
1.23
1.70
0.33
0.14
0.69
0.23
0.39
1.00
0.23
0.25
0.86
0.81
1.07
0.49
0.41
1.40
1.20
1.59
0.77
0.86
1.81
1.38
Species
Barrier
Species ´ Barrier
SEM
SEM
SEM
0.320
0.506
0.716
0.084
0.133
0.189
0.060
0.095
0.134
0.056
0.088
0.125
0.040
0.063
0.089
1987). The higher infection rate of white clover roots
than those of grass supports the proposition that clover
roots are more mycotrophic than grass roots (Haynes,
1980). Taking the 5 lm barrier as a base on which to
judge transfer of N in other treatments, about 30% more
N was transferred in the 60 lm than the 5 lm treatment.
However, this occurred irrespective of the presence of
AMF.
In the study of Haystead et al. (1988) in which they
concluded that direct hyphal transfer of N between
clover and grass occurred, a 60-lm mesh barrier was
used to allow hyphae, but not roots, to pass. However,
as found in Experiment 2 in this study, ®ne roots of
grass can penetrate a 60-lm mesh. Hence it is possible
that the greater amount of clover-derived N in grass
separated from clover by the 60-lm mesh barrier in the
mycorrhizal than the non-mycorrhizal treatment could
have been due to more N being available to the ®ne
roots of grass which had penetrated the barrier in the
clover rhizosphere in the inoculated treatment.
The absence of AMF-stimulated transfer in the 60
lm treatment in this study may have been due to the
high N transfer in the non-mycorrhizal treatment
masking any additional transfer which may have resulted from the greater root mass in the AMF treatment.
Closeness of contact between the root systems of the
legume and grass has been shown by Hamel et al. (1991)
(working with soybean and maize) to be more important
than AMF presence in N transfer.
The nutrient status of the receiver relative to that of
the donor has been considered to be a factor in determining the degree of hyphal mediated N transfer
(Bethlenfalvay et al., 1991) although Frey and Sch
uepp
(1992) recorded some AMF-mediated N transfer
158
J.B. Rogers et al. / Chemosphere 42 (2001) 153±159
Table 6
Experiment 2: details of penetration of pores in mesh barriers by roots of clover and grass
Barrier (lm)
Species
Penetration of
mesh
5
Clover
Grass
No
No
0
0
0
0
0
0
15
Clover
Grass
No
No
0
0
0
0
0
0
35
Clover
Grass
No
Yes
0
2
0
230
0
84
60
Clover
Grass
No
Yes
0
84
0
274
0
128
between berseem and maize even when maize was not
apparently N-de®cient (Barber and Martin, 1976). As
Experiment 1 progressed there was an indication that
the smallest mesh size resulted in the lowest grass yield,
suggesting that grass was relying on N from the clover
donor. Despite that, and taking account of the fact that
roots were well infected with AMF, mycorrhizal transfer
was not apparent.
Frey and Sch
uepp (1992) have used a mesh of pore
size 0.45 lm as a barrier to external hyphae of AMF. In
this study it was considered that such a small pore size
would be a hindrance to mass ¯ow and di€usion as
strong suction is required when using such membranes
for ®ltration. Rogers (1993) found no evidence of
external hyphae passing from inoculated roots through a
membrane of 5 lm pore size when they were growing
adjacent to the barrier. We are therefore convinced that
the 5-lm pore size was an e€ective barrier for hyphae.
14
C was found in the grass roots in Experiment 3,
suggesting that some form of transfer of carbon had
occurred between the two root systems. It is well known
that C is transported from shoots of AMF-infected
plants into the hyphae, e.g. in cucumber plants (Jakobsen and Rosendahl, 1990). Although there was no indication from Experiment 3 of the root systems, by
microscopy or from the autoradiographs that hyphal
links had been involved in the transfer, it is possible that
AMF external hyphae may have translocated 14 C from
the donor plants and released it into the rhizosphere of
the receivers. Alternatively, roots may have released
carbon compounds during growth (Barber and Martin,
1976; Martin, 1977) and white clover roots turn over
much faster than those of grass (Laidlaw et al., 1996).
Therefore the 14 C in grass roots may have resulted from
uptake of some of this released carbon.
Further evidence against the implication of hyphal
links in N transfer between clover and grass is the existence of partial ecological speci®city between AMF
and their host, as has been found for AMF populations
which infect white clover and perennial ryegrass (Rogers
et al., 1994; Zhu et al., 2000). In conclusion, this study
Mean root
length
Root diameter (lm)
Prior to penetration
After penetration
suggests that N transfer between white clover and
perennial ryegrass is unlikely to occur through AMF
hyphal links in agricultural soils where the opportunities
for alternative routes of transfer are likely to be well
developed.
Acknowledgements
J.B. Rogers is grateful to the Ministry of Agriculture,
Fisheries and Food for a postgraduate studentship.
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